J. Env. Bio-Sci., 2014: Vol.

28 (2): 289-292 ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

GENETIC VARIABILITY IN FIELD GROWN PLANTS REGENERATED FROM IN VITRO

CULTURED IMMATURE EMBRYOS OF BARELY (HORDEUM VULGARE L.)
Tiwari V. K., Cocking E.C. and Davey M. R.
Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, University Park,
Nottingham NG7 2RD England
email: vkt786@rediffmail.com

Received: 30-09-2014 Accepted: 29-10-2014

Embryogenic callus was successfully initiated from culturing immature embryos of cv. Dissa on CC medium fortified with
2 mg l-1 2, 4-D. Embryogenic calli were frequently produced distinguished somatic embryos on this medium. Highest frequency of
somatic embryo's germination was occurred on MS medium supplemented with 0.5 mg l-1 kinetin and 3% glucose in cv. Dissa. The
age of callus also influenced regeneration with high frequencies occurring after 6 weeks in culture. Subsequently, plant regeneration
was achieved by transferring germinated somatic embryos on hormone-free MS medium. Successfully regenerated plants from
immature embryos were grown to maturity in soil. Genetic parameters of regenerated mature plants suggested that the number
of spikes per plant, number of tillers per plant and seed yield per plant could be selected as characters for the improvement of
quantitative traits in cv. Dissa.

Tissue culture techniques can be complementary for the
conventional breeding in order to produce plants routinely in
large scale; free from diseases and pests. Regenerable callus
have been obtained from immature embryos 1,2. Plant
regeneration from somatic embryos has been reported by
several workers2-5. Genotype and medium composition effects
on organogenesis have been reported in barley 6. The response
of cultured immature embryos to different 2, 4-D concentrations
and exposure times were also reported; it is found that 2, 4-D
was an effective auxin for callus formation in gramineous
species7. In general, all above workers used different growth
regulators for callus induction and plant regeneration. We
describe here the initiation of embryogenic callus from immature
embryo-derived scutellum tissues of spring cv. Dissa of barley,
regeneration of plants via somatic embryogenesis and
organogenesis and assess genetic variability of regenerated
plants.
MATERIALS AND METHODS
20 ml of agarose-solidifed CC medium supplemented with (2
mg 1-1, 2,4-D) in 60x15mm petri-dishes 12. These petri dishes
were incubated at 25±10C in the dark condition. MS medium
was autoclaved for 20 minutes at 1200C while other media
were filter sterilized through 0.2 µm membrane before adjusting
the pH 5.8 and later combined with the gelling agent 8 g 1-1
agar.
Regeneration Media: Somatic calli of cv. Dissa were
transferred onto MS medium supplemented with 2 mg 1-1 2,
4-D 10. Cultures were maintained in the dark at 24±10C for two
weeks and then transferred to light under same culture
conditions for the enhancement in growth of plantlets. Finally,
plantlets were transferred onto MS medium (hormone free)
medium for the development of shoots under light at 24±10C.
After 2-3 weeks plants were transferred onto pots under high
humidity at 200C with fluorescent light for two weeks. After
acclimatization, plants were grown in the glasshouse until
maturity.

Materials and Explant: Embryogenic callus cultures were
initiated from immature embryos of barley (cv. Dissa). Seeds
at milk ripe to soft dough stage were taken from glasshouse
grown plants. Dehusked immature seeds were sterilized with
10% Domestos and then washed three times with sterilized
distilled water. Immature embryos (~ 0.5-0.8 mm) were excised
under stereo-microscope and 8-10 embryos were plated onto
Estimation of Fertility and Genetic Variability of
Regenerated Plants: Five plants were randomly selected for
fertility estimation at maturity. The number of fertile florets
(containing seed) and non-fertile florets (no seed set) were
scored per plant. Twenty mature plants were randomly selected
for the estimation of genetic variability. The randomized block
design was used to analyze the data4. Observations were
 Genetic Variability in Field Grown Plants (290)

Table-1. Analysis of variance, mean and standard error for quantitative characters of plants
regenerated from somatic calli derived from immature embryos of cv. Dissa

[* Significant at 0.05 probability level. **Significant at 0.01 probability level.]

Table-2. Estimates of genotypic and phenotypic variability and genetic parameters for quantitative
characters of barley plants regenerated from immature embryo-derived calli of cv. dissa

[Abbreviations: G.V = Genotypic variance; P.V = Phenotypic variance; G.V.C =Genotypic coefficient of variance;
P.C.V = Phenotypic coefficient of variation, G.A = genetic advance; C.V = Coefficient of variation]

Fig. 1. Development of plants regenerated from three week old calli of cv. Dissa on RM1+ medium to field.
(291) Tiwari, Cocking, and Davey
recorded for plant height (cm), number of tillers per plant, number
of nodes per plant, number of spikes per plant, spike length
(cm), number of florets per spike, number of seeds per spike,
seed weight per spike (g) and seed yield per plant (g). The
genotypic and phenotypic coefficients of variation (GCV and
PCV) were calculated according to the formula proposed8.
RESULTS AND DISCUSSION
The frequency of germination of somatic embryos was increased
by the addition of 0.5 mg 1-1 kinetin to MS medium with the
substitution of 3% glucose instead of 3% sucrose. Immature
embryos formed clusters of leafy structures on CC medium.
These structures lead to the formation of quatripolar callus
which developed somatic embryos after 3 weeks. In contrast,
reported that cytokinins were not sufficient for shoot development
in callus cultures, but that 1 mg 1-1 IAA and 0.05 mg 1-1
zeatin were required to induce the germination of somatic
embryos9. Other cytokinins, such as BAP, Kinetin and 2-ip,
caused browning and necrosis in barley callus. On MS medium
induced callus became brown and necrotic. Maximum
embryogenic callus development was observed on CC medium
with 2 mg 1-1 2, 4-D. The embryogenic callus was nodular,
compact and yellow in colour. Higher concentrations of 2, 4-D
was suppressed embryogenic callus formation which leads to
the formation of non-morphogenic, watery callus. In contrast,
it observed that higher concentration of 2, 4-D (5 mg 1-1) was
used for the initiation of cultures10.
Plant regeneration from immature embryo-derived callus
Optimum embryo germination frequencies and optimum
numbers of green shoots produced per callus were occurred
on MS medium supplemented with 0.5 mg 1-1 kinetin [RM1+
medium]. The germination of somatic embryos also occurred
if cultures were left for 5 weeks on agarose-solidified initiation
medium without subculture. The germinated embryos produced
albino plantlets with leaflets and roots. On RM1+ medium;
somatic embryos germinated and produced green plantlets
after 5 weeks of culture when 3 week old calli were cultured on
RM1+ medium (Fig. 1). In another study, the regenerable callus
was obtained when explants were cultured on MS medium
containing IAA or 2, 4-D by several workers 11. The plant
regeneration was obtained by organogenesis when calli were
transferring to MS medium lacking hormones for roots and
shoots development and then transferred in pots for further
development. Plants reached to maturity in 17 weeks after
transfer to compost. Plant regeneration from immature embryo-
derived callus of Hordeum spontaneum and H. Bulbosum were
occurred on a medium; containing IAA and zeatin12. Hence, it
is observed that in vitro regeneration of plant is strongly
genotype dependent. Similar results were reported by and
have been found that genotype, size, age of explants, growth
condition and effects of media composition on the callus
induction6,7,13,14. They reported that six week old calli showed
a high percentage of plantlet formation when they were
transferred onto the regeneration medium containing 0.5 mg
1-1 kinetin. In contrast, the high frequency of plantlet
regeneration occurred; when 3 to 4 week old calli with somatic
embryos were transferred onto the plant regeneration medium
containing 1.0 mg 1-1 IAA and 0.05 mg 1-1 zeatin9. They
found that embryogenic callus cultures of barley could be
maintained for 8 months, but that totipotency generally declined
after one month of culture. In contrast, plant regeneration
reported after 4 months of culture14.
Genetic variability of plants regenerated from immature
embryo-derived callus of cv. Dissa. Analysis of variance (Table- 1)
showed that, of 9 characters examined8, the number of tillers
per plant and the seed yield (g) per plant varied significantly
(P < 0.01) within the population of plants regenerated from
immature embryo-derived callus. This indicated that high
variability exist in these characters. Other characters, such
as plant height (cm), number of nodes per plant, number
spikes, spike length (cm), number of florets per spike, number
of seeds per spike and seed weight per spike (g), showed
that no statistically significant variation (P < 0.05) (Table-2).
The genotypic coefficient of variation was calculated as ranging
from 4.3 to 146.3 and phenotypic coefficient of variation ranged
from 14.9 to 220.6 (Table-2). This indicated that phenotypic
coefficient of variation was higher than genotypic coefficient
of variation for all of the characters. It indicates that phenotypic
variability was more than genotypic expression of all traits
under study. The number of tillers per plant and seed yield per
plant (g) showed high heritability coupled with low genetic
advance (Table-2). Moderate heritability was observed in the
number of spikes per plant and spike length (cm) and low
heritability in number of nodes per plant. In both cases,
heritability was coupled with low genetic advance.
Developmental traits, such as number of spikes per plant,
seed yield per plant (g), number of tillers per plant showed
substantially higher genetic coefficients of variation with high
 Genetic Variability in Field Grown Plants
heritability (except for number of spikes per plant) and low
genetic advance indicating that selection for these traits would 3.
be effective in future selection programmes. A wide variability
in plant height, number of fertile tillers, number of non-fertile 4.
tillers and fertility, with a range of 2, 4-D concentrations (1-4
mg l-1) and there were no differences in the frequency of plant
regeneration at 1-3 mg 1-1 2,4-D but 4 mg 1-1 decreased the
5.
plant regeneration frequency when calli were transferred for 2-
4 weeks onto B5 medium containing these 2,4-D
6.
concentrations7. It was suggested that where the maintenance
of genetic stability is usually important, it would be advisable
not to use a high level of 2, 4-D. They found in their experiment 7.
that plants were not produced from immature barley embryos
cultured either on various 2,4-D concentrations for a short time 8.
(4 days) or at low 2,4-D concentration (0.1 mg 1-1) for different 9.
times. It seems that an intervening callus phase is sometimes
necessary for plant production. Possibly, the callus plays a 10.
nurse role in the development of embryogenic tissue.
The present investigation revealed the induction of embryogenic 11.
callus which lead to the plant regeneration via somatic
12.
embryogenesis and organogenesis at large scale and
13.
regenerated plants successfully transferred in the field for
maturity. Thus, in vitro regenerated plants escaped from many
diseases also. 14.
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